59results about How to "Improve expression rate" patented technology

The invention belongs to the field of immunotherapy and relates to a universal CAR-T cell and a preparation method and application thereof. In the universal CAR-T cell, the functions of a T cell antigen receptor (TCR) and major histocompatibility complexes (MHC I and MHC II) in the T cell are inhibited while multi-gene knockout is performed; a gene encoding the TCR includes TRAC and / or TRBC; genesencoding the major histocompatibility complexes include HLA-A, B2MH and CIITA. The universal CAR-T cell can target relevant markers of specific tumors and inactivate the functions of the TCR and theMHC on the cell surface, can reduce immunological rejection caused by allogeneic cell therapy and safely and effectively remove tumor cells in the diseased human body, is not affected by the disease or a treatment mode of a patient in use and can be prepared at any time, treatment can be provided at the optimum time, and treatment effectiveness is ensured.

An expression system for producing a protein in a filamentous fungus, consisting of a) a host organism selected from the species Trametes and Polyporus and b) a DNS vector containing a selection marker gene. This selection marker gene code is a protein which allows the selection of positive transformants after the transformation of the host organism and which is selected from the group of: antibiotics-resistant genes coding for proteins which eliminate the growth-inhibitory effect of antibiotics against which the host organism is not resistant. Genes coding proteins which are capable of a color-causing reaction; and genes complementing a genetic defect of the host organism (auxotrophy), the expression of the selection marker gene is controlled by at least one active genetic regulation element in the host organism, and c) a DNS vector containing a gene which codes the protein to be produced. The expression of this gene and optionally, the secretion of the protein so produced are controlled by an active genetic regulation element in the host organism. The DNS vector containing a section marker gene and the DNS vector containing the gene which codes the protein to be produced can also be in the form of one DNS vector.

The invention relates to a human peripheral blood lymphocytes culture medium and the application thereof. The human peripheral blood lymphocytes culture medium of the invention comprises: RPMI1640 liquid culture medium 77-81.9%, bovine blood 10-14.9%, PHA 4-9%, non-sulfated glycosaminoglycan 2-5%, CPPs 2-5% and Double-resist 0.05-0.1%. The culture medium provided in the invention achieves a high lymphocyte conversion rate, a high lymphocyte split index and a good reagent stability; can obtain a human chromosome figure which can provide a firm basis to chromosome core type analysis and clinic diagnosis.

The invention provides a method for inducing mesenchymal stem cells (MSCs) into chondrocytes. The method comprises the following steps of: obtaining MSCs and the chondrocytes; inoculating the chondrocytes into a chondrocyte culture solution to be cultured; inoculating MSCs in an inserted cell culture dish, placing the inserted cell culture dish in the chondrocyte culture solution again, and replacing the chondrocyte culture solution into a chondrocyte induced differentiation culture medium for co-culture. By adopting the induction method, MSCs and the chondrocytes can simulate an in-vivo environment to produce a synergy, so that induced MSCs are differentiated into chondrocyte factors to play a role of a synergistic effect for inducing factors with an extracellular matrix secreted by the chondrocytes, so as to obviously shorten the time for inducing MSCs to differentiate into chondrocytes, improve the proliferation rate of the chondrocytes, and simultaneously enhance the expression rate and the expression quantity of type II collagens which induce the differentiated cells and the secretion of glycosaminoglycan (GAG).

The invention is about the Cephalosporium acremonium transformation leaded by the plasmid Ti of the Agrobacterium tumefaciens. Compared to the 1-4 transformants it can get above 100 transformants from the 107 receptor cells. So the transforming efficient has improved greatly.

The invention relates to an induction method for promoting differentiation of human umbilical cord mesenchymal stem cells into chondrocytes, which comprises the following steps of: obtaining an umbilical cord tissue block, a primary culturing stem cells, subculturingstem cells and inducing stem cells differentiate to chondrocytes, wherein the induction for differentiation of stem cellsinto chondrocytes comprises the following steps of: resuspending human umbilical cord mesenchymal stem cells subcultured to a seventh generation by adopting a serum-free culture medium to prepare P7 generation stem cell suspension; inoculating P7 generation stem cell suspension into the serum-free culture medium containing a cartilage differentiation inducer for continuous culture; replacing the serum-free culture medium containing the cartilage differentiation inducer in a half amount every three days; obtaining chondrocytes after 14 days.The induction method can differentiate human umbilical cord mesenchymal stem cells into chondrocytes,which has important significance in repairing human cartilage tissues and treating bone injuries.

The invention discloses a composition for efficiently inducing and amplifying human peripheral blood killer immune cells, a kit and a culture method of the immune cells. The composition is prepared from interleukin-1 beta, interleukin-6, prostaglandin E2, interferon gamma, a CD3 monoclonal antibody, a CD28 monoclonal antibody, interleukin-2, a tumor necrosis factor-alpha, a granulocyte macrophage stimulating factor, interleukin-1 alpha, interleukin-4 and a PD-1 antibody or a CTLA-4 antibody. The composition provided by the invention can effectively improve the multiplication capacity of killer T autoimmune cells and the tumor cell killing capacity of the killer T autoimmune cells.

The invention relates to an expression vector and a preparation method and application thereof, in particular to a multi-copy integrated expression vector and a preparation method and application thereof in expression of bovine lactoferrin, and belongs to the technical field of gene engineering. The nucleotide sequence of the multi-copy integrated expression vector is expressed as SEQ ID No.1. In the multi-copy integrated expression vector, YIPlac204 plasmid is used as a skeleton, PGK is used as a promoter, G418 resistance is used as a selection marker, and rDNA is used as an integrated site; and the vector is a yeast multi-copy integrated expression vector suitable for industrialized production.

The invention discloses a construction method for a gene recombination baculovirus expression vector and relates to a construction method for an HA-VP2 gene recombination baculovirus expression vector. The invention aims to solve the problem that the expression rate of a traditional baculovirus-mediated exogenous gene in a Chinese Hamster Ovary (CHO) cell is low. The method comprises the following steps of: I-, performing polymerase chain reaction (PCR) amplification by taking pMD18-T-VP2 as a template to obtain a target fragment, and performing TA cloning and vector conjugation on the target fragment to obtain pTZF-HA-VP2; II-, respectively performing double-enzyme digestion on nine plasmids and the pTZF-HA-VP2, and connecting target fragments obtained after the enzyme digestion is ended to obtain nine recombination transfer vectors; III-, respectively transforming the nine recombination transfer vectors into competent cells, and extracting to obtain rBac-TZF-X; and IV-, performing transfection on the sf9 cells by the rBac-TZF-X to obtain rBV-TZF-X. According to the construction method for the HA-VP2 gene recombination baculovirus expression vector, the expression rate of the baculovirus-mediated exogenous gene in the CHO cell can be increased through adding a regulatory element WPRE.

The invention discloses an efficient expression vector of an antibody. The efficient expression vector contains resistance genes and two target gene binding sites for binding the genes, the two target gene binding sites can be respectively combined with heavy chain genes and light chain genes of antibody molecules, the two target gene binding sites are respectively positioned in two expression units in the efficient expression vector and respectively form a heavy chain expression unit and a light chain expression unit of the antibody, and each expression unit also comprises a strong expression inductivity promoter positioned on the upstream of each target gene binding site. The efficient expression vector can simultaneously express the heavy chain and the light chain of the antibody, solve the problem about imbalanced expression of the heavy chain and the light chain of the antibody in the prior art, increase the expression rate of antibody protein and improve the screening effectiveness of subsequent monoclonal antibody cell strains.

The invention discloses plasmid vector mediated gene therapy recombinant vectors of the same category. The invention relates to the field of bio-pharmaceuticals and particularly relates to a recombinant vector capable of efficiently expressing a human hepatocyte growth factor and the application thereof. The invention further relates to the application of a medicine containing a constructed enhanced signal peptide for improving protein expression and a composition thereof in preparation of medicines for treating lower extremital arterial ischemic disease.

The invention relates to a preparation method of an antibacterial peptide cecropin feed additive, belonging to the technical field of gene engineering. The preparation method of the antibacterial peptide cecropin feed additive comprises the following steps of: (1) carrying out enzyme cutting to an antibacterial peptide cecropin gene and a multicopy integration expression vector respectively by Not I, after connecting by DNA ligase, converting E.coli TOP10 bacterial strains, coating an LB (Luria-Bertani) flat plate, carrying out enzyme cutting verification by the Not I, and selecting and naming a transformant inversely inserted into a cecA1 fragment as pYIP-cecA1; (2) carrying out enzyme cutting to the pYIP-cecA1 prepared in step (1) by using Hpa I, recovering big fragments, adding the pYIP-cecA1 into 80 muL of yeast electricity transformation competent cells, carrying out electric shock on a mixture, and screening the mixture by the YEPD (Yeast Extract Peptone Dex) flat plate of G418 to obtain the yeast cell for expressing the cecropin; and (3) culturing the yeast cell for expressing the cecropin, which is prepared in step (2), and drying to obtain the cecropin feed additive. According to the preparation method, the production cost of the cecropin can be lowered, and a good foundation is laid for popularizing the cecropin to serve as the feed additive to be antibiotics substitute for feeds.

A gene for the non-fusion recombination of human alkaline fibroblast growth factor, its carrier and prokaryotic expression system, said non-fusion recombined human alkaline fibroblast growth factor and the application of said factor in preparing medicines for treating nervous system injury are disclosed.

The invention relates to a preparation method for a human umbilical cord mesenchymal stem cell preparation used for conventional anti-aging treatment. The method includes the following steps: acquisition of an umbilical cord tissue block, primary culture of stem cells, subculture of the stem cells, cryopreservation of the stem cells, recovery and culture of the stem cells, and preparation of the human umbilical cord mesenchymal stem cell preparation; and the step of the preparation of the human umbilical cord mesenchymal stem cell preparation includes the following steps: resuspension subculture is carried out to Pe-generation human umbilical cord mesenchymal stem cell by employing 5% of glucose injection, after a Pe-generation human umbilical cord mesenchymal stem cell suspension is uniform, 5% of the glucose injection is supplemented, the concentration of the Pe-generation human umbilical cord mesenchymal stem cell is adjusted to 4*10<4>-6*10<4> / mL to obtain the human umbilical cordmesenchymal stem cell preparation; and wherein e=6, 7, 8, 9, and 10. The preparation method improves reservation of human umbilical cord mesenchymal stem cells, and ensures the survival rate, the proliferation capability and the specificity of the human umbilical cord mesenchymal stem cells.

The invention relates to a method of producing peptides restructured by polypeptide with bioactivity, including following steps: a. Synthesize genes with respect to coding area of peptides amino acids artificially; b Genes gained from step a fuse with 3íõside of bacteria thioredoxin genes on the proper expression carriers, among which there are enterokinase or fibrin ferment recognition site dots; c Expression carriers of the coding genes in fusion proteins gained from step b invert proper bacillus colis to get gene engineering bacterias; d said gene engineering bacteria is handled through fermentation, extraction, chromatography, purification steps to prepare peptides- thioredoxin fusion proteins; e said fusion proteins gained from step d is handled by dissection of enterokinase or fibrin ferment, separation, purification to acquire restructed peptides.

The present invention pertains to the field of recombinant protein production. Novel peptides derived from the extracellular region of a glycophorin protein are provided which enhance the expression rate of proteins or peptides of interest when expressed as fusion protein together with said novel peptides.

The invention discloses a fermentation medium for preparing recombination IL-1ra (Interleukin-1 Receptor Antagonist) and a fermentation method thereof. The fermentation method comprises the following step of: inoculating recombination strains on the fermentation medium for fermentation cultivation to obtain a fermentation broth, namely obtaining a recombination IL-1ra. The recombination strains are recombination escherichia coli obtained by guiding genes shown in sequence 1 in a sequence table into escherichia coli, and the guided genes which are shown in the sequence 1 in the sequence table are guided into the escherichia coli via the following recombination expression vectors: inserting the genes shown in the sequence 1 of the sequences into multiple clone sites of a PET-30a vector to obtain the recombination expression vectors. The method has good repeatability; the growth of the strains is fast so as to greatly shorten the period of fermentation; the yield of the strain is high, which can meet the high-density fermentation requirements, and target proteins has high expression rate and good solubility.

The invention relates to an induction method for promoting differentiation of human umbilical cord mesenchymal stem cells to osteoblasts, which comprises the following steps of: acquiring umbilical cord tissue blocks, carrying out primary culture on the stem cells, carrying out subculture on the stem cells and induction differentiation of the stem cells to chondrocytes, wherein the induction of the differentiation of the stem cells to the chondrocytes comprises the following steps of: carrying out resuspension subculture on a serum-free culture medium until Pe generation human umbilical cord mesenchymal stem cells are prepared into a Pe generation human umbilical cord mesenchymal stem cell suspension solution; the Pe generation human umbilical cord mesenchymal stem cell suspension solutionis cultured in serum-free medium containing an osteogenic differentiation inducer, the serum-free medium containing the osteogenic differentiation inducer is replaced every three and a half days, after the induction is carried out for 14 days, osteoblasts are obtained; wherein e=6, 7, 8, 9, 10. The method can differentiate human umbilical cord mesenchymal stem cells into osteoblasts, and has great significance in repairing human bone tissues and treating bone injuries.

The invention relates to a human peripheral blood lymphocytes culture medium and the application thereof. The human peripheral blood lymphocytes culture medium of the invention comprises: RPMI1640 liquid culture medium 77-81.9%, bovine blood 10-14.9%, PHA 4-9%, non-sulfated glycosaminoglycan 2-5%, CPPs 2-5% and Double-resist 0.05-0.1%. The culture medium provided in the invention achieves a high lymphocyte conversion rate, a high lymphocyte split index and a good reagent stability; can obtain a human chromosome figure which can provide a firm basis to chromosome core type analysis and clinic diagnosis.

The invention provides a lentiviral vector expression system for realizing polygene transformation by splitting a single selection marker, and comprises the lentiviral vector expression system, a construction method thereof and application in the polygene transformation. By use of the lentiviral vector expression system provided by the invention, transformation of a plurality of target genes can be realized by using the single selection marker, and the packaging rate of the expression system and the expression rate of the target genes can be improved in various cells.

The invention belongs to the technical field of stem cells, and particularly relates to an amniotic mesenchymal stem cell culture medium and a method for culturing amniotic mesenchymal stem cells. The amniotic mesenchymal stem cell culture medium provided by the invention comprises a basic fibroblast growth factor, a platelet derived cell growth factor, panax japlcus var saponin and lentinan. Through reasonable compatibility of the components, cell proliferation can be promoted, the expression rate and the cell purity of the amniotic mesenchymal stem cells are improved, and a sufficient number of stem cells are obtained within a relatively short time; the amniotic mesenchymal stem cell culture medium is applicable to two-dimensional or three-dimensional culture of the amniotic mesenchymal stem cells, so that research and development needs are met.

A method of screening cell clones expressing a high yield of a polypeptide of interest is provided. The method employs the consecutive use of fluorescence activated cell sorting followed by colony picking based selection of cell clones with high expression rates and high proliferation rates. Furthermore, the invention pertains to a method of producing a polypeptide of interest using cells obtained by the described screening method.

The invention relates to the technical field of bioengineering, and particularly relates to a method and a medium for amplifying bone-marrow mesenchymal stem cells and application. The method for amplifying bone-marrow mesenchymal stem cells comprises the steps of treating the bone-marrow mesenchymal stem cells with bFGF and VEGF to amplify the bone-marrow mesenchymal stem cells. The bFGF and VEGFare added to the medium. The bone-marrow mesenchymal stem cells cultured by the method provided by the invention can increase the proliferation ability of the cells, and maintain the biological characteristics and functions of the cells, and also can increase the expression rate of the cell surface molecule CD105, thereby being useful for transplant treatment of acute myocardial infarction, chronic myocardial infarction, and ischemic heart disease caused by cardiac failure.

The invention belongs to the technical field of stem cells and particularly relates to a culture method for enhancing differentiation from mesenchymal stem cells to chondrocytes. The method includes:(1) extracting chondrocytes and inoculating the chondrocytes on a cell culture liquid for cultivation; (2) extracting the mesenchymal stem cells and inoculating the mesenchymal stem cells on an insertcell culture dish; (3) placing the insert cell culture dish and the chondrocytes, cultured in the step (1), together in a cell inducing differentiation culture medium for coculture, wherein the cellinducing differentiation culture medium containing human bone morphogenetic protein 2 and growth hormone HGH. In the invention, the chondrocytes and mesenchymal stem cells are cocultured on the inducing culture medium, wherein in-vivo environment is simulated under induction by the culture medium on the two cells, thus initiating synergistic effect, increasing proliferation rate of the chondrocytes and meanwhile simultaneously increasing the expression rate and quantity of collagen in the cells after the inducing differentiation.

The present invention provides a respiratory syncytial virus (RSV) recombinant fusion protein (F protein) in which a polymerization domain derived from a foreign protein is bound to the C terminal of a fusion protein (F protein) lacking a transmembrane domain of a wild-type respiratory syncytial virus (RSV) fusion protein (F protein). The recombinant fusion protein of the present invention is soluble and can retain an F protein trimer. Excellent immune-inducing effects can be expected from the recombinant fusion protein of the present invention, and the vaccine composition containing the same.

The invention relates to the technical field of adoptive T cell immunotherapy, in particular to a T cell enriching method and an application thereof to adoptive T cell therapy. The enriching method comprises the following steps: culturing peripheral blood mononuclear cells (PBMCs) for 1-4 hours, then removing adherent cells, and then separating T cells from the remaining cells by using magnetic beads. The method can effectively improve the purity of the T cells, the yield of the T cells is high, and the operation is simple. According to the method disclosed by the invention, the expression rate of a chimeric antigen receptor (CAR) is improved by changing a virus infection method, and the operation risk is reduced.

The invention relates to a method for culturing mesenchymal stem cells without serum, and belongs to the field of stem cells. The method includes the steps that mononuclear cells are separated from marrows, and the mesenchymal stem cells with positive cell phenotypes CD73, CD90 and CD105 are obtained through multiplication culture. A serum-free medium is used during culturing, by optimizing components of the medium and the proportion of all the components, pollution to foreign proteins and exogenous microorganisms is avoided effectively, a stem cell form and stem cell features obtained throughculturing are good, and the proliferation rate of cells and positive expression rate of the mesencymal stem cells are increased remarkably. Through the method, new purposes are provided for large-scale preparation of the mesenchymal stem cells and cell transplantation treatment.

The invention relates to methods and system for amplifying bone marrow mesenchymal stem cells, and a computer readable medium. The method includes performing induction culturing on bone marrow mesenchymal stem cells for 1-3 days under conditions that temperature is at 36-38 DEG C, the volume concentration of N2 is 91-93%, the volume concentration of air is 1%, the volume concentration of CO2 is 4.5-5.5% and the volume concentration of O2 is 1.5-2.5%; and performing continuous culturing on the induction-cultured bone marrow mesenchymal stem cells under the conditions that the temperature is at36-38 DEG C, the volume concentration of CO2 is 4.5-5.5% and the volume concentration of O2 is 15-25% so as to amplify the bone marrow mesenchymal stem cells. The method can rapidly amplify the bone marrow mesenchymal stem cells and obtain the bone marrow mesenchymal stem cells with the best proliferative capacity, biological activity and endothelial differentiation function.

The invention relates to the field of stem cells, and discloses a cryopreservation solution and a cryopreservation method for mesenchymal stem cells. The cryopreservation solution for mesenchymal stem cells comprises a serum-free medium, DMSO and dextran-40, wherein the concentration of the DMSO is 3-7 v / v %, and in g / mL, the concentration of the dextran-40 is 1-3 w / v %. After the cryopreservation solution for mesenchymal stem cells disclosed by the invention is used, the survival rate of cryopreserved cells is significantly increased, and good stem cell characteristics are kept. In the cryopreservation method for mesenchymal stem cells disclosed by the invention, after cells are resuspended by using the cryopreservation solution disclosed by the invention and the cell density is adjusted, the cells are cryopreserved. After cells cryopreserved by using the cryopreservation method for mesenchymal stem cells disclosed by the invention recover, the survival rate of the cells is obviously increased, the cell reproductive capacity is also superior to that in conventional cryopreservation methods, and specific surface markers of mesenchymal stem cells also do not have obvious drop, and have a high expression rate.

The invention provides a method for inducing mesenchymal stem cells (MSCs) into chondrocytes. The method comprises the following steps of: obtaining MSCs and the chondrocytes; inoculating the chondrocytes into a chondrocyte culture solution to be cultured; inoculating MSCs in an inserted cell culture dish, placing the inserted cell culture dish in the chondrocyte culture solution again, and replacing the chondrocyte culture solution into a chondrocyte induced differentiation culture medium for co-culture. By adopting the induction method, MSCs and the chondrocytes can simulate an in-vivo environment to produce a synergy, so that induced MSCs are differentiated into chondrocyte factors to play a role of a synergistic effect for inducing factors with an extracellular matrix secreted by the chondrocytes, so as to obviously shorten the time for inducing MSCs to differentiate into chondrocytes, improve the proliferation rate of the chondrocytes, and simultaneously enhance the expression rate and the expression quantity of type II collagens which induce the differentiated cells and the secretion of glycosaminoglycan (GAG).